This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
Improvements in diode laser, fiber optic, and data acquisition technologies are enabling increased use of Raman spectroscopic techniques for both in-lab and in-situ water analysis. In this disclosure “in-situ” is used to describe a situation in which the measurement or action is or can be performed directly at the source of a solution or sample in the field. Thus in this disclosure “in-situ” can be used interchangeably with each of “in the field”, “on-site, “in-line” and “in-flow”. Aqueous media encountered in the natural environment often contain suspended solids that can interfere with spectroscopic measurements. Removal of these solids, for example via filtration, can have adverse effects on the extent to which subsequent measurements are representative of actual field conditions under which the aqueous media are collected. Turbidity is a measure of the loss of optical transparency of a medium resulting from the presence of suspended solids or other interfering matter, which can limit the overall sensitivity of optical spectroscopic methods and make it challenging to perform quantitative analysis.
Turbidity is measured by various methods known to those skilled in the art and is typically measured in NTUs (Nephelometric Turbidity Units). NTUs provide a standardized measure of the extent to which white light is scattered at an angle of 90° from the direction of the incident beam by particles suspended in a liquid relative to the same effect observed in a standard solution containing the polymer reaction byproduct of hydrazine sulfate and hexamethylenetetramine, in accordance with Environmental Protection Agency (EPA) Method 180.1, and are a standard measure of water quality in environmental science.
The adverse influence of turbidity on optical spectroscopic measurements is recognized in many fields. In oceanographic investigations, the challenges posed by turbidity for in-situ spectroscopic analyses have been acknowledged for several decades. Studies have indicated that turbidity limited the effectiveness of studies of petroleum films, chlorophyll distribution, and sea water temperature and salinity performed using airborne laser sounding. In a similar domain, a reduction in the Raman backscatter of water with increasing turbidity in airborne examinations of water optical transmission was observed. It has been reported that turbidity can adversely impact potential to measure low concentrations of natural and hazardous chemicals in the ocean. In the context of environmental science, several researchers have indicated that the presence of suspended particulates in open fresh water and groundwater can lead to scattering, absorption, and displacement of an unknown sample volume that would otherwise be interrogated by optical sources in a spectroscopic system and can thus lead to inaccurate measurements of in-situ chemical concentrations.
Several attempts have been made to apply correction to Raman observations affected by turbidity. Some researchers desire to see “through” the turbid medium to perform spectroscopic analysis on underlying layers of material. This is the case in many biological scenarios and studies of pharmaceuticals. In these contexts, researchers have developed techniques to 1) work with the limited amount of unaffected light returned from a turbid sample (e.g., confocal microscopy or multiphoton microscopy, often at considerable complexity and expense, 2) invert the scattered return, sometimes employing sophisticated time gating, with tradeoffs in resolution peer “through” turbid layers via what has been termed interferometric focusing with the limitation of a need for a priori access to the target focal plane, 4)) employ non-linear chemometricor or bioinformatic methods to address non-analyte specific signal variances stemming from turbidity through techniques such as support vector regression, after considerable experimental trials or work with simulated samples.
Some studies focused on studying the bulk turbid material itself—that is the matrix responsible for generating the turbidity effect in the sample—which in many applications is granular or powdery in nature (e.g., bulk active pharmaceutical ingredient analysis). Here again time-resolved scattering observations can be employed to discern the source of scattered return as a function of the probable scattered photon path length, or a spatial offset between the incident illumination source and the collected return can be employed to reassemble an image from diffusely scattered light. In addition, researchers have highlighted the merits of employing a large interrogation spot size and making use of reflecting mirrors or diffuse reflectors to enhance Raman returns when the scattering medium is of analytical interest. Still other researchers perform analyses of liquid (water) or tissue samples and wish to study the chemical composition of the fluid or tissue via quantitative analysis of the medium in which the turbid inducing constituents are suspended (more narrowly, the Raman scatterers within the turbid medium). In these contexts, differing methods have been employed for different target materials, with notable variations in approach between the laboratory and the field.
For analysis of biological tissues in the laboratory, where turbidity correction is arguably the most advanced, corrections were initiated in the context of fluorescence studies. Here researchers recognized that fluorescent and diffusely reflected photons behave in a similar manner in turbid media, and methods were derived to obtain what is termed “intrinsic fluorescence”—that is the fluorescence emanating from direct excitation incidence on the target—from either the ratio of measured fluorescence to diffuse reflectance at a given emission wavelength, or through interpretation of photon migration models of concomitantly measured fluorescence and reflectance.
A similar line of logic was employed by researchers in the development of intrinsic Raman spectroscopy (IRS) and turbidity-corrected Raman spectroscopy (TCRS) which are both based on the photon migration approach and employ alternate acquisition of Raman and diffuse reflectance spectra to obtain corrected Raman observations. The IRS method employs a Monte Carlo calibration model based on extensive analysis of phantom media representative of target constituents, as well as accurate knowledge of constituent Raman scattering coefficients. The TCRS method overcomes these limitations through a theoretical link between the observed Raman spectrum, the diffuse reflectance spectrum, and the turbidity-corrected Raman spectrum. The method, however, requires complex calibration to obtain an instrument specific constant, determination of the average photon path length in the turbid media under investigation, and estimation of the diffusely reflected light at the Raman excitation frequency. Some studies have suggested turbidity corrections based on the relationship between Raman return and sample reflectance, but distinguish the contributions of absorption and scattering, and employ a Monte Carlo simulation to obtain a corrected Raman signal from an inferred combination of the target material absorption coefficient and reduced scattering coefficient. Although complex, and challenging to implement in a natural field setting, the breakdown of the influences of turbidity on Raman observations provided by these model-based corrections reveal that for quantitative characterization of turbid media, turbidity-induced variations in sampling volume—that is the turbidity inducing constituents occupy a fraction of the interrogated sample volume—often become dominant over other forms of spectral distortion (absorption and scattering).
This premise has historically been exploited in the natural environment where turbidity corrections have been primarily linked to direct or indirect measurements of sample turbidity that provide an indication of combined scattering, absorption, and volume reduction effects. Some researchers corrected measurements of fluorescent tracers in model aquifers for turbidity related signal attenuation through cyclic excitation of target media and a known fluorescence reference positioned on the far side of a flow through sampling vessel, enabling real time correction for signal amplitude, with the known drawback of potential interference from fluorophores within the target media. Others put forward a simple groundwater fluorometer signal intensity correction based on an empirically derived relationship between sample turbidity level and related changes in scattered excitation energy. Some researchers corrected UV/VIS absorption measurements of paper mill wastewater for turbidity by a theoretical relation linking turbidity to light attenuation based on an assumption of the diameter of the turbidity inducing particles, and a spectral correction relating changes in scattering at any given wavelength to turbid particle diameter. Others demonstrated the potential to correct observations of fluorescing dissolved organic matter for turbidity effects through instrument specific calibration of excitation light absorption as a function of dissolved organic matter content in target media, and empirically derived relations between field derived turbidity levels and fluorescence signal attenuation, noting the need for periodic collection of water samples and measurement of filtered samples in the laboratory to assess the appropriate magnitude of the correction.
As outlined above prior attempts to correct for the adverse effects of turbidity rely on one or more of the following: pass-through optical observations, a priori modeling of the test medium, significant excitation penetration into a sample, the opportunity to excite and collect optical energy at displaced locations, and/or large area optical excitation and/or collection, or optical behavior characterized by the Beer-Lambert law. Generally, few of these conditions, if any, can be met in a typical in-situ field monitoring scenario. Thus a need exists for methods of correcting for turbidity effects in Raman observations in practical monitoring scenarios in the field without the need for “pass through” optical observations, without the need for a priori modeling thus enabling corrections in “real time” thereby accounting for in-situ changes, and without the need to deeply penetrate the sample with radiation or analyze large areas, thus enabling corrections in “real time” to account for in-situ changes, and enabling targeted, single-sided optical observations.